Enzyme-labeled antigens are widely used in solid-phase immunoassays, and have been proposed for use in homogeneous immunoassays. In a solid-phase assay, the enzyme-labeled antigen is designed to complete with an analyte in solution for binding to anti-analyte binding molecules carried on the surface of a solid support. After the initial competitive-inhibition binding reaction, the solid support is washed and assayed for enzyme activity. The amount of enzyme activity associated with the support is inversely proportional to the amount of analyte present in the original assay reaction mixture. This assay system has the advantage of greater sensitivity over solid-phase immunoassay systems employing antigen/fluorophore or antigen/chromophore conjugates. At the same time, the system avoids the problems and expense involved in measuring radionuclides encountered in solid-phase radioimmune assays (RIA), where the analyte-competing antigen is radiolabeled.
In a homogeneous enzyme immunoassay, the enzyme-labeled antigen is designed typically to compete with an analtye for binding to an anti-analyte antibody which is free in the reaction mixture. The relative spatial arrangement of antigen and enzyme is such that antibody binding to the antigen produces a measurable, normally inhibitory effect on the enzyme. Therefore, the presence and/or concentration of analyte in solution can be measured as a change in enzyme activity in the assay mixture.
Heretofore, numerous methods for attaching enzyme labels to antigens to form conjugates for use in enzyme immunoassays have been described (references 1, 2). In general, these methods employ chemical coupling methods, such as periodate oxidation and glutaraldehyde coupling, or cross-linking agents such as o-phenylene dimaleimide, dihydroxysuccinimide, or soluble dicarbodimides. A serious limitation of chemical coupling methods is the difficulty in achieving consistent and reproducible antigen/enzyme coupling in terms of (a) the site(s) of antigen attachment to the enzyme, (b) the number of antigens attached to each enzyme molecules, and (c) the extent of loss of enzymz activity or change in immunological activity of the antigen following coupling. Because of the difficulty in controlling these variables, the manufacturer of the assay kit typically must carry out quality control tests on each batch of enzyme-labeled antigen that is produced. Even with the quality controls, it is difficult to predict how variations in antigen binding to an enzyme will affect the enzyme on storage. Also, a homogeneous-type assay may be quite sensitive to variations in the number of antigens bound to the enzyme and/or their sites of attachment, to an extent that many antigen/enzyme reagent for use in a homogeneous assay canot be produced practically by chemical coupling methods.
U.S. Pat. No. 4,378,428 describes a homogeneous assay system composed of a peptide antigen fused to a small N-terminal fragment of .beta.-galactosidase antigen antibody, and a complementary C-terminal .beta.-galactosidase enzyme fragment, both components produced by recombinant DNA techniques. Binding of an anti-antigen antibody to the fused protein interfers with the ability of the N-terminal portion of the enzyme to form an active enzyme complex with a relatively large C-terminal enzyme fragment. This complementary enzyme system is somewhat expensive to manufacture, and is subject to greater assay variations related to enzyme concentration and stability effects than immunoassays systems employing a single enzyme species. Moreover, the system would have limited uses in a solid-phase immunoassay, since the amount of enzyme bound to the solid support could not be assayed directly.
It is therefore an object of the present invention to provide a fused enzyme/peptide protein and methods and systems for producing and using the protein, which overcome above-discussed limitations in prior art enzyme immunoassay reagents.
The invention includes a fused protein reagent for use in an enzyme immunoassay. The protein includes an enzymatically active .beta.-galactosidase fused, at its C-terminus, to an immunologically active peptide. The active peptide is preferably derived from an immunologically active region of a peptide hormone, a serum protein, or other suitable polypeptide analyte.
The invention further includes a plasmid for use in constructing a fused protein gene. The plasmid contains a complete-sequence .beta.-galactosidase gene which terminates at a selected restriction endonuclease site. In producing the fused protein, a nucleotide coding for the immunologically active peptide is inserted in the plasmid at the selected restriction site, and a transfected host which produces the desired fused protein is identified by the presence of .beta.-galactosidase activity and immunospecific reaction with an anti-peptide antibody.
The fused protein may be used in a solid-phase enzyme immunoassay based on competitive inhibition between an analyte and the fused protein for binding immunospecifically to a solid support. In another embodiment the fused protein forms part of a homogeneous assay system in which antibody binding to the fused protein modulates the protein's enzyme activity.
These and other objects and features of the present invention will be come more fully apparent when the following detailed description of the invention is read in conjunction with the accompanying drawings.